in my luciferase assay, i transfect SV40 as internal control together with the promoter region which is in the PGL3 vector and my genes of interest, however the reading value showed that my genes of interest have the reasonable reading, but the SV40 seems no expressed at all. I am sure the SV40 plasmid is OK, because it was working in my colleague's experiment. I transfect 2ng in each well of the 96 well plate. I do not know why? So if i want to optimize the transfection amount of SV40, may I transfect a range of SV40 alone in each well? Then how about the substrate and stop buffer, do I need to add both as I did in the normal co-transfection and see the second reading? or I just add one? which?
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#2
dpo
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Posted 29 September 2009 - 04:55 AM
which cells are you using? I've heard (not from my own experience) that the SV40 promoter is not active in IMR32 or Colo320DM cells.
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bry512
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Posted 29 September 2009 - 05:37 PM
It is a mouse epithelial cell line. The cells should be ok, my previous colleagues also use this cell line transfected with SV40.
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Posted 06 October 2009 - 03:02 AM
I use lipofectamine 2000, and follow the protocol.
